Funds are requested for the purchase of a dedicated system to perform multiphoton intravital microscopy (MPIVM), which will be installed as a core instrument in the CBR Institute for Biomedical Research. The new MPIVM system will constitute an expansion of the Institute's existing multiphoton/confocal microscopy core facility. User demand for access to the established facility far exceeds its capacity, which has caused month-long waiting lists and severely restricts investigators' ability to make progress in NIH-funded research projects. The most rapid growth in usage of the existing setup has been in the area of intravital microscopy whereby three-dimensional time-lapse movies are generated to study intra- and extravascular events in lymph nodes, bone marrow and the brain of anesthetized mice. While investigator-initiated preliminary work in this area has been remarkably productive, possibility for further progress is limited due to lack of access and also because the current core facility was designed to exhibit maximum versatility and is not specifically optimized for MPIVM imaging. The proposed new system will thus meet an urgent need of Major Users who will use MPIVM to pursue the following NIH-funded research projects: 1.) Imaging of antigen trafficking in vivo (M.C. Carroll); 2.) Visualization of immune cell traffic in bone marrow (IB. Mazo); 3.) Visualization of MHC class II transport in dendritic cells (H. Ploegh); 4.) T cell migration and differentiation in lymph nodes (U.H. von Andrian); and 5.) Assessment of blood-brain barrier function in vivo (D.D. Wagner). The CBR Institute for Biomedical Research has committed institutional funds for construction/renovation of a dedicated room and to purchase a new Olympus BX51 fluorescence microscope and a vibration-isolated table system, which are integral components of the MPIVM setup. CBR funds will also support a full-time Optical Imaging Facility Manager and a stand-alone workstation for image analysis and data processing. Funds are requested to procure a MaiTai broadband mode-locked Ti:Sapphire laser (Spectra-Physics) and a multiphoton-optimized Radiance 2100 laser scanning system from Bio-Rad.